Methods for detecting cancer cells by using humanized antibodies which bind specifically to FB5 antigen

ABSTRACT

The invention provides for the production of several humanized murine antibodies specific for the antigen FB5, which is recognized by the murine antibody FB5. The FB5 antigen is expressed on the luminal surface of vascular endothelial cells of a wide range of malignant tumors. The invention also provides for numerous polynucleotide encoding humanized FB5 specific antibodies, expression vectors for producing humanized FB5 specific antibodies, and host cells for the recombinant production of the humanized antibodies. The invention also provides methods for detecting cancerous cells (in vitro and in vivo) using humanized FB5 specific antibodies. Additionally, the invention provides methods of treating cancer using FB5 specific antibodies.

RELATED APPLICATIONS

This application is a divisional of Ser. No. 09/013,872, filed on Jan.27, 1998, which is a divisional of Ser. No. 08/657,012, filed on May 20,1996 U.S. Pat. No. 5,811,522, which is a continuation of Ser. No.08/207,778, filed on Mar. 8, 1994, abandoned.

FIELD OF THE INVENTION

The present invention is related to the field of molecular biology, andmore particularly to humanized antibodies.

BACKGROUND

The present invention relates to the generation, by recombinant DNAmethods, of novel recombinant immunoglobulins specific for the human FB5(endosialin) cancer antigen. The invention also discloses methods forthe production of these recombinant antibodies, for the diagnosis andtreatment of certain human cancers.

Transformation of a normal cell to a malignant cell is often accompaniedby a change in the expression of cell surface antigens. These differentphenotypes can be detected using monoclonal antibodies specific for suchantigens. In this way, different cancer cells can be detected andcharacterized (Lloyd, K. O. (1983) “Human Tumour Antigens: Detection andCharacterization with Monoclonal Antibodies” in R. B. Herberman, ed.,Basic and Clinical Tumour Immunology, pp 159-214, Martinus Nijhoff,Boston). These cell surface antigens are appropriate targets for tumourimmunotherapy and diagnosis.

Of particular value for the diagnosis and therapy of a broad range ofcancers would be the identification of an antigen associated with abroad range of cancers. Tumour stromas are potential sites for thelocation of such antigens. One such antigen (F19) is expressed on thesurface of reactive stromal fibroblasts associated with >90% ofepithelial cancers (Garin-Chesa, P. et al. (1990) Proc. Natl Acad. Sci.87, 7235-7239; Rettig, W. J. et al. (1988) Proc. Natl Acad. Sci. 85,3110-3114). In clinical trials, a monoclonal antibody specific for theF19 antigen accumulated at tumour sites successfully locating hepaticmetastases from colorectal carcinomas (Welt, S. et al. (1992) Proc. Am.Assoc. Cancer Res. 33, 319). This illustrates the diagnostic potentialof monoclonal antibodies specific for tumour stromal antigens. Anothertumour stromal antigen (FB5 or ‘endosialin’) has been identified andpartially characterized (Rettig, W. J. et al. (1992) Proc. Natl Acad.Sci. 89, 10832-10836). A murine monoclonal antibody (mAbFB5) has beenraised against the FB5 antigen. This antibody has been used to show thatthe FB5 antigen is expressed on the luminal surface of vascularendothelial cells of a wide range of malignant tumours. Specifically, inimmunohistochemical analyses of vascular endothelial cells of humantumours, FB5 expression was found in 26 of 36 carcinomas, 18 of 25neuroectodermal tumours and 41 of 61 sarcomas. In contrast, it could notbe detected in any of a wide range of normal adult tissues includingtissues of the following organ systems : breast, cardiovascular,connective tissues, digestive tract, endocrine, haematopoietic,lymphoid, reproductive, skin and urinary. Similary, FB5 was notexpressed in cultured human malignant cells (excepting a subset ofsarcomas), and stromal fibroblasts of only a small proportion ofepithelial cancers exhibited FB5 expression.

The specificity of the FB5 murine antibody makes it a powerful tool forthe detection of human cancers in vitro. For a number of reasons thelocation of the FB5 antigen on the luminal surface of tumour vascularendothelial cells makes the antigen an ideal target for tumourimmunotherapy and diagnosis in vivo. Firstly, a wide range of cancertypes may be diagnosed and treated by the FB5 antibody (or antibodyconjugate). Secondly, the endothelial cell surface is readily accessibleto antibodies that are circulating in the blood stream. Thirdly,antibody-targeted destruction of tumour blood vessels could lead towidespread necrosis in solid tumours. Finally, on binding toFB5—expressing cells the mAbFB5 is rapidly internalized raising thepossibility that the antibody could be used for the specific delivery ofcytoxic agents for the destruction of tumour blood vessels. However, thein vivo use of murine antibodies as agents for the diagnosis andtreatment of human diseases in severely curtailed by a number offactors. Specifically, the human body recognises murine antibodies asforeign. This recognition of the murine antibodies can elicit a humananti-mouse antibody (HAMA) response (Schroff, R. et al. (1985) CancerRes. 45, 879-885) which results in rapid clearance of the antibody fromthe circulation. Furthermore, the Fc portion of a murine antibody is notas efficacious as the human Fc at stimulating human complement orcell-mediated cytotoxicity. For the in vivo use of murine antibodies indiagnosis and therapy, these problems must be circumvented.

EP120694 (Celltech) and EP125023 (Genentech) disclose the development of‘chimaeric’ antibodies using recombinant DNA methods. Such antibodiescomprise the variable regions from one species (eg mouse) and theconstant regions from another species (eg human).

Such chimaeric antibodies would have the advantage that they retain thespecificity of the murine antibody but can also stimulate human Fcdependent complement fixation and cell-mediated cytotoxicity. However,the murine variable regions can still elicit a HAMA response(Bruggemann, M. et al. (1989) J. Exp. Med. 170, 2153-2157) therebylimiting the value of chimaeric antibodies as diagnostic and therapeuticagents.

British Patent Application Number GB2188638A (Winter) discloses aprocess whereby recombinant antibodies can be generated by substitutionof only the variable region CDRs of one antibody with those fromanother. Typically, this ‘CDR-grafting’ technology has been applied tothe generation of recombinant, pharmaceutical antibodies consisting ofmurine CDRs, human variable region frameworks and human constant regions(eg Riechmann, L. et al, (1988) Nature, 332, 323-327). Such ‘reshaped’or ‘humanized’ antibodies have less murine content than chimaericantibodies and retain the human constant regions necessary for thestimulation of human Fc dependent effector functions. In consequence,CDR grafted antibodies are less likely than chimaeric antibodies toevoke a HAMA response when administered to humans, their half-life incirculation should approach that of natural human antibodies and theirdiagnostic and therapeutic value is enhanced.

In practice, for the generation of efficacious humanized antibodiesretaining the specificity of the original murine antibody, it is notusually sufficient simply to substitute CDRs. In addition there is arequirement for the inclusion of a small number of critical murineantibody residues in the human variable region. The identity of theseresidues depends on the structure of both the original murine antibodyand the acceptor human antibody. British Patent Application Number9019812.8 discloses a method for identifying a minimal number ofsubstitutions of foreign residues sufficient to promote efficaciousantigen binding.

The present invention provides novel, humanized monoclonal antibodiesspecific for the human FB5 cancer antigen. This has been achieved by theconversion of the murine FB5 monoclonal antibody to humanized antibodiesby utilising CDR-grafting technologies. The invention also providesmethods for the production of these humanized antibodies to be used inthe diagnosis and treatment of certain human cancers. Prior to the workof the inventors, it was not known that FB5 or any other non-humanantibody specific for the the FB5 antigen could be humanized so as toretain useful binding specificity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Shows the DNA sequence and corresponding amino acid sequence ofthe murine FB5 heavy chain variable region (VH). The CDRs are boxed.Underlined nucleotides and amino acid residues are derived from theoligonucleotide primers used. A backslash is used to indicate the resultobtained by the use of consensus primers.

FIG. 2 Shows the DNA sequence and corresponding amino acid sequence ofthe murine FB5 light chain variable region (VK). The CDRs are boxed.Underlined nucleotides and amino acid residues are derived from theoligonucleotide primers used.

FIG. 3 Shows the vector pSVgpt for the expression of chimaeric orhumanized heavy chains in mammalian cells.

FIG. 4 Shows the vector pSVhyg for the expression of chimaeric orhumanized light chains in mammalian cells.

FIGS. 5-10 These figure provide graphical data of ELISA resultsdemonstrating the binding properties of humanized FB5 specificantibodies.

SUMMARY OF THE INVENTION

One aspect of the invention is to provide humanized antibodies specificfor the FB5 antigen.

Another aspect of the invention is to provide polynucleotides encodinghumanized antibodies specific for the FB5 antigens. Various expressionvectors comprising polynucleotides encoding humanized FB5 antibodiesjoined to promoter sequences are also provided. Similarly, anotheraspect of the invention is host cells transformed with expressionvectors for the expression of humanized FB5 specific antibodies.

Another aspect of the invention is to provide humanized anti-FB5antibodies that are labeled with a detectable label or a therapeuticlabel.

Another aspect of the invention is to provide methods for treatingand/or diagnosing cancer by administering a composition comprising ahumanized FB5 specific antibody. One method of detecting cancer cellsinvolves the steps of administering a labeled antibody (detectablelabel) to a patient and subsequently detecting where in the body thelabeled antibody has bound.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

As used herein, the term “humanized” antibody refers to a molecule thathas its CDRs (complementarily determining regions) derived from anon-human species immunoglobulin and the remainder of the antibodymolecule derived mainly from a human immunoglobulin. The term “antibody”as used herein, unless indicated otherwise, is used broadly to refer toboth antibody molecules and a variety of antibody derived molecules.Such antibody derived molecules comprise at least one variable region(either a heavy chain of light chain variable region) and includemolecules such as Fab fragments, Fab′ fragments, F(ab′)₂ fragments, Fdfragments, Fabc fragments, Fd fragments, Fabc fragments, Sc antibodies(single chain antibodies), diabodies, individual antibody light chains,individual antibody heavy chains, chimeric fusions between antibodychains and other molecules, and the like.

The term “conventional molecular biology methods” refers to techniquesfor manipulating polynucleotides that are well known to the person ofordinary skill in the art of molecular biology. Examples of such wellknown techniques can be found in Molecular Cloning: A Laboratory Manual2nd Edition, Sambrook et al, Cold Spring Harbor, N.Y. (1989). Examplesof conventional molecular biology techniques include, but are notlimited to, in vitro ligation, restriction endonuclease digestion, PCR,cellular transformation, hybridization, electrophoresis, DNA sequencing,cell culture, and the like.

The term “variable region” as used herein in reference to immunoglobulinmolecules has the ordinary meaning given to the term by the person ofordinary skill in the act of immunology. Both antibody heavy chains andantibody light chains may be divided into a “variable region” and a“constant region”. The point of division between a variable region and aheavy region may readily be determined by the person of ordinary skillin the art by reference to standard texts describing antibody structure,e.g., Kabat et al “Sequences of Proteins of Immunological Interest: 5thEdition” U.S. Department of Health and Human Services, U.S. GovernmentPrinting Office (1991).

The present invention provides humanized antibody molecules specific forFB5 antigen in which at least parts of the CDRs of the heavy and/orlight chain variable regions of a human antibody (the receptor antibody)have been substituted by analogous parts of CDRs of a murine monoclonalantibody and the humanized antibody can specifically bind to the same asthe FB5 antibody. In a preferred embodiment of the subject invention,the CDR regions of the humanized FB5 specific antibody are derived fromthe murine antibody FB5. Some of the the humanized antibodies describedherein contain some alterations of the acceptor antibody, i.e., human,heavy and/or light chain variable domain framework regions that arenecessary for retaining binding specificity of the donor monoclonalantibody. In other words, the framework region of some embodiments thehumanized antibodies described herein does not necessarily consist ofthe precise amino acid sequence of the framework region of a naturaloccurring human antibody variable region, but contains varioussubstitutions that improve the binding properties of a humanizedantibody region that is specific for the same target as the murine FB5specific antibody. A minimal number of substitutions are made to theframework region in order to avoid large-scale introductions ofnon-human framework residues and to ensure minimal immunogenicity of thehumanized antibody in humans. The donor monoclonal antibody of thepresent invention is the FB5 murine antibody, which is specific for thehuman FB5 cancer antigen.

The humanized antibodies of the present invention include completeantibody molecules having full length heavy and light chains, or anyfragment thereof, such as the Fab or (Fab′)₂ fragments, a heavy chainand light chain dimer, or any minimal fragment thereof such as a Fv, anSCA (single chain antibody), and the like, specific for the FB5 antigenmolecule.

In addition to providing for humanized FB5 specific antibodies, thesubject invention provides for polynucleotides encoding humanized FB5specific antibodies. The subject polynucleotides may have a wide varietyof sequences because of the degeneracy of the genetic code. A person ofordinary skill in the art may readily change a given polynucleotidesequence encoding a humanized FB5 specific antibody into a differentpolynucleotide encoding the same humanized FB5 specific antibodyembodiment. The polynucleotide sequence encoding the antibody may bevaried to take into account factors affecting expression such as codonfrequency, RNA secondary structure, and the like.

The humanized antibodies of the subject invention may be produced by avariety of methods useful for the production of polypeptides, e.g. invitro synthesis, recombinant DNA production, and the like. Preferably,the humanized antibodies are produced by recombinant DNA technology.

The humanized FB5 specific antibodies of the invention may be producedusing recombinant immunoglobulin expression technology. The recombinantproduction of immunoglobulin molecules, including humanized antibodiesare described in U.S. Pat. No. 4,816,397 (Boss et al), U.S. Pat. No.4,816,567 (Cabilly et al) U.K. patent GB 2,188,638 (Winter et al), andU.K. patent GB 2,209,757. Techniques for the recombinant expression ofimmunoglobulins, including humanized immunoglobulins, can also be found,among other places in Goeddel et al, Gene Expression Technology Methodsin Enzymology Vol. 185 Academic Press (1991), and Borreback, AntibodyEngineering, W. H. Freeman (1992). Additional information concerning thegeneration, design and expression of recombinant antibodies can be foundin Mayforth, Designing Antibodies, Academic Press, San Diego (1993).

The recombinant humanized anti-FB5 antibodies of the invention may beproduced by the following process or other recombinant proteinexpression methods:

a. Constructing, by conventional molecular biology methods, anexpression vector comprising an operon that encodes an antibody heavychain in which the CDRs and a minimal portion of the variable regionframework that are required to retain donor antibody binding specificityare derived from a non-human immunoglobulin, such as the murine FB5monoclonal antibody, and the remainder of the antibody is derived from ahuman immunoglobulin, thereby producing a vector for the expression of ahumanized antibody heavy chain.

b. Constructing, by conventional molecular biology methods, anexpression vector comprising an operon that encodes an antibody lightchain in which the CDRs and a minimal portion of the variable regionframework that are required to retain donor antibody binding specificityare derived from a non-human immunoglobulin, such as the murine FB5monoclonal antibody, and the remainder of the antibody is derived from ahuman immunoglobulin, thereby producing a vector for the expression ofhumanized antibody light chain.

c. Transferring the expression vectors to a host cell by conventionalmolecular biology methods to produce a transfected host cell for theexpression of humanized anti-FB5 antibodies.

d. Culturing the transfected cell by conventional cell culturetechniques so as to produce humanized anti-FB5 antibodies.

Host cells may be cotransfected with two expression vectors of theinvention, the first vector containing an operon encoding a heavy chainderived polypeptide and the second containing an operon encoding a lightchain derived polypeptide. The two vectors may contain differentselectable markers but, with the exception of the heavy and light chaincoding sequences, are preferably identical. This procedure provides forequal expression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA or both.

The host cell used to express the recombinant antibody of the inventionmay be either a bacterial cell such as Escherichia coli, or preferably aeukaryotic cell. Preferably a mammalian cell such as a chinese hamsterovary cell, may be used. The choice of expression vector is dependentupon the choice of host cell, and may be selected so as to have thedesired expression and regulatory characteristics in the selected hostcell.

The general methods for construction of the vector of the invention,transfection of cells to produce the host cell of the invention, cultureof cells to produce the antibody of the invention are all conventionalmolecular biology methods. Likewise, once produced, the recombinantantibodies of the invention may be purified by standard procedures ofthe art, including cross-flow filtration, ammonium sulphateprecipitation, affinity column chromatography, gel electrophoresis andthe like.

The humanized FB5 specific antibodies of the present invention may beused in conjunction with, or attached to other antibodies (or partsthereof) such as human or humanized monoclonal antibodies. These otherantibodies may be reactive with other markers (epitopes) characteristicfor the disease against which the antibodies of the invention aredirected or may have different specificities chosen, for example, torecruit molecules or cells of the human immune system to the diseasedcells. The antibodies of the invention (or parts thereof) may beadministered with such antibodies (or parts thereof) as separatelyadministered compositions or as a single composition with the two agentslinked by conventional chemical or by molecular biological methods.Additionally the diagnostic and therapeutic value of the antibodies ofthe invention may be augmented by labelling the humanized antibodieswith labels that produce a detectable signal (either in vitro or invivo) or with a label having a therapeutic property. Some labels, e.g.radionucleotides may produce a detectable signal and have a therapeuticproperty. Examples of radionuclide labels include ¹²⁵I, ¹³¹I, ¹⁴C.Examples of other detectable labels include a fluorescent chromophoresuch as fluorescein, phycobiliprotein or tetraethyl rhodamine forfluorescence microscopy, an enzyme which produces a fluorescent orcolored product for detection by fluorescence, absorbance, visible coloror agglutination, which produces an electron dense product fordemonstration by electron microscopy; or an electron dense molecule suchas ferritin, peroxidase or gold beads for direct or indirect electronmicroscopic visualization. Labels having therapeutic properties includedrugs for the treatment of cancer, such as methotrexate and the like.

The subject invention also provides for a variety of methods fortreating and/or detecting cancer cells. These methods involve theadministration to of humanized FB5 specific antibodies, either labelledor unlabelled, to a patient. One method of detecting cancer cells in ahuman involves the step of administering a labeled humanized FB5specific antibody (labelled with a detectable label) to a human andsubsequently detecting bound labeled antibody by the presence of thelabel.

The recombinant antibodies of this invention may also be used for theselection and/or isolation of human monoclonal antibodies, and thedesign and synthesis of peptide or non-peptide compounds (mimetics)which would be useful for the same diagnostic and therapeuticapplications as the antibodies (e.g. Saragovi et al., (1991) Science253:792-795).

When the humanized FB5 specific antibodies of the invention are used invitro, the antibodies are typically administered in a compositioncomprising a pharmaceutical carrier. A pharmaceutical carrier can be anycompatible, non-toxic substance suitable for delivery of the monoclonalantibodies to the patient, Sterile water, alcohol, fats, waxes, andinert solids may be included in the carrier. Pharmaceutically acceptedadjuvants (buffering agents, dispersing agent) may also be incorporatedinto the pharmaceutical composition.

The humanized antibodies compositions of the invention may beadministered to a patient in a variety of ways. Preferably, thepharmaceutical compositions may be administered parenterally, i.e.,subcutaneously, intramuscularly or intravenously. Thus, this inventionprovides compositions for parenteral administration which comprise asolution of the human monoclonal antibody or a cocktail thereofdissolved in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers can be used, e.g., water, buffered water,0.4% saline, 0.3% glycine and the like. These solutions are sterile andgenerally free of particulate matter. These compositions may besterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate, etc. The concentration of antibody inthese formulations can vary widely, e.g., from less than about 0.5%,usually at or at least about 1% to as much as 15 or 20% by weight andwill be selected primarily based on fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

Actual methods for preparing parenterally administrable compositions andadjustments necessary for administration to subjects will be known orapparent to those skilled in the art and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th Ed., MackPublishing Company, Easton, Pa. (1980), which is incorporated herein byreference.

The subject invention provide numerous humanized antibodies specific forthe FB5 antigen based on the discovery that the CDR regions of themurine monoclonal antibody could be spliced into a human acceptorframework so as to produce a humanized recombinant antibody specific forthe FB5 antigen. Preferred humanized FB5 specific antibodies containadditional change in the framework region (or in other regions) toincreasing binding for FB5 antigen. Particularly preferred embodimentsof the invention are the exemplified humanized antibody molecules thathave superior binding properties for FB5.

The following examples are offered by way of illustration of theinvention, and should not be interpreted as a limitation of theinvention.

EXAMPLES

In the following examples all necessary restriction and modificationenzymes, plasmids and other reagents and materials were obtained fromcommercial sources unless otherwise indicated.

Unless otherwise indicated, all general recombinant DNA methodology wasperformed as described in “Molecular Cloning, A Laboratory Manual”(1989) Eds J. Sambrook et al., published by Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

In the following examples these abbreviations may be employed:

dCTP deoxycytidine triphosphate dATP deoxyadenosine triphosphate dGTPdeoxyguanosine triphosphate dTTP deoxythymidine triphosphate DTTdithiothreitol C cytosine A adenine G guanine T thymine PBS phosphatebuffered saline PBSB phosphate buffered saline containing 0.5% (w/v)bovine serum albumin PBST phosphate buffered saline containing 0.05%(v/v) Tween-20

Example 1 Production of Humanized Antibodies Specific for the FB5Antigen

The source of the donor CDRs used to prepare these recombinantantibodies was a murine monoclonal antibody, mAbFB5, which is specificfor the FB5 antigen of certain human cancers (Rettig, W. J. et al.(1992) Proc. Natl Acad. Sci. 89, 10832-10836). The FB5 monoclonalantibody was produced by immunisation of (BALB/c x A) F₁ mice with humanfibroblasts and subsequent production and screening of hybridoma cells.Cytoplasmic RNA was prepared from the mAb FB5 hybridoma cell line by themethod of Favoloro, J. et al., (1980), Methods in Enzymology, 65,718-749). cDNA was synthesised using Ig variable region primers asfollows: for the Ig heavy chain variable region (VH), the primer CG2aFOR(5′ GGAAGCTTAGACCGATGGGGCTGTTGTT TTG 3′) (SEQ ID NO:1); for the lightchain variable region (VK), the primer CK2FOR (5′GGAAGCTTGAAGATGGATACAGTTGGTGCAGC 3′) (SEQ ID NO:2). cDNA synthesisreactions consisted of 4 μg RNA, 25 pmol CG2aFOR or CK2FOR, 250 μM eachof dATP, dCTP, dGTP and dTTP, 100 mM TrisHCl pH8.3, 140 mM KCl, 10 mMDTT, 10 mM MgCl₂ and 31.5 units of RNase inhibitor (Pharmacia, MiltonKeynes, U.K.) in a total volume of 50 μl. Samples were heated to 70° C.for 10 minutes (min) then slowly cooled to 42° C. over a period of 30min. 100 units of Moloney Murine Leukaemia virus (M-MLV) reversetranscriptase (Life Technologies Ltd, Paisley, U.K.) was added andincubation at 42° C. continued for 1 hour.

VH and VK cDNAs were then amplified using the polymerase chain reaction(PCR) as described by Saiki, R. K. et al., (1988), Science, 239,487-491. The primers used were:

CG2aFOR (5′ GGAAGCTTAGACCGATGGGGCTGTTGTT TTG 3′)  (SEQ ID NO:b 1)

CK2FOR (5′ GGAAGCTTGAAGATGGATACAGTTGGTGCAGC 3′)  (SEQ ID NO:2)

VH1BACK (5′ AGGTSMARCTGCAGSAGTCWGG 3′) (SEQ ID NO:3)

SK2BACK (5′ ACTAGTCGACATGGRCTTHMAGRTGSAG 3′)  (SEQ ID NO:4)

where M=C or A, H=not G, R=A or G, S=C or G, and W=A or T. Such primersand their use in the PCR amplification of mouse Ig DNA are described byOrlandi, R. et al., (1989), Proc. Natl Acad. Sci. U.S.A., 86, 3833-3837.For PCR amplification of VH, 5 μl RNA/cDNA hybrid was mixed with 25 pmolCG2aFOR and VHIBACK primers. For PCR amplification of VK, 5 μl RNA/cDNAhybrid was mixed with 25 pmol CK2FOR and SK2BACK primers. To thesemixtures was added 200 μM each of dATP, dCTP, dGTP and dTTP, 67 mMTrisHCl pH8.8, 17 mM (NH₄)₂SO₄, 10 mM MgCl₂, 0.02%(w/v) gelatin and 2.5units of AmpliTaq DNA polymerase (Perkin Elmer Ltd, Beaconsfield, U.K.)in a total volume of 50 μl. These were then subjected to 25 thermalcycles of PCR at 94° C., 30s; 50° C., 40s; 72° C., 30s; ending with 5min at 72° C. For cloning and sequencing, amplified DNA was purified byelectrophoresis in a low melting point agarose gel and by Elutip-dcolumn chromatography (Schleicher and Schuell, Dussel, Germany).Amplified VH DNA was cut with HindIII and PstI and cloned into M13mp18or M13mp19 cut with HindIII and PstI (Life Technologies Ltd, Paisley,U.K.). Amplified VK DNA was cut with HindIII and SalI and cloned intoHindIII and SalI cut M13mp18 or M13mp19 (Life Technologies Ltd, Paisley,U.K,).

The resulting clones were sequenced by the dideoxy method (Sanger, F. etal., (1977), Proc. Natl Acad. Sci. U.S.A., 74, 5463-5467) usingSequenase (United States Biochemical, Cleveland, Ohio, U.S.A.). The DNAand protein sequences of the FB5 VH and VK domains are shown in FIGS. 1and 2. The location of the CDRs was determined with reference to Kabat,E. A. et al. (1987) “Sequences of Protein of Immunological Interest”,U.S. Department of Health and Human Services, US Government PrintingOffice, and utilising computer assisted alignment with other VH and VKsequences.

The transfer of the murine CDRs to human frameworks was achieved byoligonucleotide site-directed mutagenesis, based on the method ofNakamye, K. and Eckstein, F. (1986) Nucleic Acids Res. 14, 9679-9698.The human framework regions chosen to receive the transplanted CDRs wereNEWM and REI for the heavy and light chains respectively. The structuresof these proteins have been solved crystallographically. The templatesfor mutagenesis were human framework region genes containing irrelevantCDRs and consisted of synthetic DNAs cloned into M13 phage (Riechmann,L. et al. (1988) Nature, 332, 323-327). The oligonucleotides used were:

NEWM VH

VHCDR1 5′ CGTCCAGGTGGCTGTCTCACCCAGTGTATAACATAGTCAGTGAA GGTGTAGCCAGACGCGGTGCAGGTCAGGCTC 3′  (SEQ ID NO: 5)

VHCDR2 5′TTGTCACTCTGCCCTTGAACTTCTGGTTGTAGGTAGTATCATCATCATAAGGATTAATATATCCAATCCACTCAAG 3′  (SEQ ID NO:6)

VHCDR3 5′CCTGAGGAGACGGTGACGAGACTCCCTTGGCCCCAGTAGTCCATAGAGTAGTCAAAGTAACCATCATAGGAATTCCCCCTTCTTGC ACAATAATAG 3′  (SEQ IDNO:7)

REI VK

VKCDR1 5′GGAGCCTTACCTGGGGTCTGCTGGTACCAGGCTACAGCAGTACCCACATTCTGGCTGGCTCTACAGGTG 3′  (SEQ ID NO:8)

VKCDR2 5′CTGCTTGGCACACCAGTGTACCGATTCGATGCCGAGTAGATCAG CAGC 3′  (SEQ IDNO:9)

VKCDR3 5′CTACTCACGTTTGATTTGCACCTTGGTCCCTTGGCCGAACGTGTACATGGGATAGTTGGTATATTGCTGGCAGTAGTAGGTGG 3′  (SEQ ID NO:10)

A number of additional, murine residues were introduced into thevariable region frameworks by u sing a separate oligonucleotide or byextension of the CDR primers. Specifically:

NEWM V(24) changed to A (NEWM VHCDR1 oligonucleotide) NEWM S(27) changedto Y (NEWM VHCDR1 oligonucleotide) NEWM S(30) changed to T (NEWM VHCDR1oligonucleotide)

 NEWM K(75), QFS(77-79), A(85) to S,TAY,E (olignucleotide: 5′CGCGGTGTCCTCGGCTGTCACGCTGCTGAGTCTCAGGTAGGCTGTGTTG GAGCTGGTGTCTACC3′)  (SEQ ID NO:1)

These residues that have been changed are believed to be important forretaining original antigen specificity. Although the invention is notdependent upon any particular explanation for the results obtained bymaking the additional residue changes, some possible explanations fortheir significance are as follows:

The change of residues NEWM V(24) to the smaller A facilitates theaccommodation of the heterlogous CDR1 loop. The NEWM S(27) to Y changewas made because S(27) is an unusual residue in subgroup II human heavychains (Riechmann et al. (1988) Nature 332, 323-327). Amino acidsVH(27-30), are residues of the ‘vernier zones’ as defined by Foote andWinter (Foote, J. and Winter G. (1992) J. Mol. Biol. 224, 487-499. Thesezones are important for adjusting CDR structures to promote antigenbinding. This explanation accounts for the changes NEWM S(27) to Y andNEWM S(30) to T. For site directed mutagenesis the VH and VKoligonucleotides encoding the murine CDRs and NEWM K,QFS,A change werephosphorylated with T4 Kinase (Life Technologies Ltd, Paisley, U.K.). A25 fold molar excess of each of the three VH (plus NEWM K,QFS,A primer)or VK primers were added to 0.5 μg of appropriate VH or VK singlestranded template DNA in M13 (NEWM VH : M13VHPCR1; REI : M13VKPCR2) in40 mM Tris HCl pH7.5, 20 mM MgCl₂, 50 mM NaCl and annealed by heating to90°0 C. for a few minutes and slowly cooling to 37° C. The annealed DNAwas extended with 2.5 units of T7 DNA polymerase (cloned, United StatesBiochemical, Cleveland, Ohio, U.S.A.) in a reaction mixture containing0.5 units of T4 DNA ligase (Life Technologies Ltd, Paisley, U.K.), 0.25mM of each of dATP, dGTP, dTTP, and dCTP (Pharmacia, Milton Keynes,U.K.), 40 mM Tris HCl pH7.5, 20 mM MgCl₂, 50 mM NaCl, 6.5 mM DTT and 1mM ATP in a total volume of 30 μl. The mixture was incubated at roomtemperature for 1 h. A 1 μaliquot of this extension/ligation mixture wasthen used in an asymmetric PCR for the specific amplification of thenewly synthesized strand. The reaction contained 1 μl extension/ligationmixture, 250 μM of each of dATP, dGTP, dTTP and dCTP, 67 mM Tris HClpH8.8, 17 mM (NH₄)₂SO₄, 10 mM MgCl₂, 0.02% (w/v) gelatin, 2.5 Units ofAmpliTaq DNA polymerase and 25 pmol of appropriate oligonucleotideprimer (5′ AACAGCTATGACCATG 3′ (SEQ ID NO:12) for NEWM VH; 5′CTCTCTCAGGGCCAGGCGGTGA 3′ (SEQ ID NO:13) for REI VK) in a total volumeof 50 μl. The reaction mixtures were subjected to 30 thermal cycles ofPCR at 94° C., 30 s; 55° C., 30 s; 72° C., 1 min ending with 72° C., 5min. The newly synthesized strand was then amplified by adding 20 pmolof appropriate oligonucleotide primer (5′ GTAAAACGACGGCCAGT 3′ (SEQ IDNO:14) for NEWM VH and 5′ GCGGGCCTCTTCGCTATTACGC 3′ (SEQ ID NO:15) forREI VK) and adjusting the reaction mixture to include a further 5 nmolsof each of dATP, dGTP, dTTP and dCTP and 2.5 Units of AmpliTaq. Thereactions were subjected to a further 20 PCR cycles as above. Theamplified VH and VK DNAs were purified from 1.5% w/v low melting pointagarose gels by elutip-d column chromatography. Purified DNA wasdigested with HindIII and BamHI plus RsaI (for VHs) or BstXI (for VKs)(all reaction enzymes from Life Technologies Ltd, Paisley, U.K.). Thereis an RsaI site in the parental VHPCR1 and a BstXI site in the parentalVKPCR2 but these sites are deleted during mutagenesis. These digestionstherefore select for newly synthesized DNA. The HindIII/BamHI digestedVH and VK DNAs were ligated into HindIII/BamHI cut M13mp18 or M13mp19(both from Pharmacia, Milton Keynes, U.K.) and transformed intocompetant E. coli TG1 (Amersham International plc, Amersham, U.K.).Single stranded DNA was prepared from individual ‘plaques’ and sequencedby the dideoxy method using Sequenase (United States Biochemical,Cleveland, Ohio, U.S.A.) according to Manufacturer's instructions.Triple CDR mutants were identified in this way and selected forconstruction of VH and VK expression vectors.

The expression vectors for the humanized VH and VK genes, pSVqpt andpSVhyg are shown in FIGS. 3 and 4. The humanized VH genes, together withthe immunoglobulin heavy chain promoter, appropriate splice sites andsignal peptide sequences were excised from the M13 clones with HindIIIand BamHI and ligated into the heavy chain expression vector, pSVqpt.This vector contains the murine heavy chain immunoglobulin enhancer, theqpt gene under control of the SV40 promoter/enhancer for selection inmammalian cells, the human IgG1 constant region domain and sequences forreplication and selection in E. coli. The humanized VK gene was clonedinto the light chain expression vector pSVhyg in the same way. Allfeatures of pSVhyg are the same as in pSVgpt except that the gpt gene isreplaced by the gene for hygromycin resistance (hyg) and a human kappaconstant region is included instead of the IgG1 constant region.

For transfection into mammalian cells 10 μg of the heavy chainexpression vector DNA and 20 μg of the light chain vector DNA werelinearized by digestion with PvuI (Life Technologies Ltd, Paisley,U.K.), coprecipitated with ethanol and redissolved in 20 μl of water.The recipient cell line was NSO, a non-immunoglobulin producing mousemyeloma, obtained from the European collection of Animal Cell Cultures,Porton, U.K., ECAC No. 85110505 cells were grown in Dulbecco's ModifiedEagle's Medium supplemented with 10% foetal calf serum and antibiotics(DMEM) (Life Technologies Ltd, Paisley, U.K.). Approximately 10⁷ NSOcells were harvested by centrifugation and resuspended in 0.5 ml DMEM,the digested DNA was added and the cells transferred to a cuvette andplaced on ice for 5 min. A single pulse of 170 volts, 960μ farads wasadministered (Genepulser, BioRad, Richmond, Calif., U.S.A.). After afurther 30 min on ice the cells were replaced in a flask in 20 ml DMEMand allowed to recover for 24 hours. After this time the cells weredistributed into a 24 well plate in selective medium (DMEM with 0.8μg/ml mycophenolic acid and 250 μg/ml xanthine). After 3 to 4 days themedium was changed for fresh selective medium. Colonies of transfectedcells were visible after 10 to 14 days.

The production of human antibody in the wells containing transfectedclones was measured by ELISA. Capture antibody, goat anti-human IgG,gamma chain specific (Sera-Lab Ltd, Crawley Down, U.K.) was diluted to 5μg/ml in 50 mM carbonate buffer pH9.6, and used to coat polystyreneELISA plates (Dynatech Immulon 1), 200 μl per well, overnight at 4° C.After washing 3 times with PBST, 50-100 μl of the culture medium to bescreened was added to the wells and incubated at 37° C. for 60 min. Thewells were washed again with PBST and the reporter antibody,peroxidase-conjugated goat anti-human IgG, gamma chain specific(Sera-Lab Ltd, Crawley Down, U.K.) or peroxidase-conjugated goatanti-human kappa chain (Sera-Lab Ltd, Crawley Down, U.K) was added at100 ng per well and the plate incubated for a further 60 min. The platewas washed as before then the colour was developed. Substrate buffer wasprepared by mixing 100 mm citric acid and 100 mM disodium hydrogenphosphate to pH5.0. 25 mg of o-phenylenediamine was dissolved in 50 mland 5 μl of 30% hydrogen peroxide added just before use. 200 μl wasdispensed per well and incubated at room temperature in the dark. Thereaction was stopped by addition of 50 μl per well of 12.5% sulphuricacid and the absorbances were read at 492 nm.

Positive cell clones were expanded for antibody purification. For thefinal expansion to production volume the cells were diluted in DMEMcontaining 10% IgG-free fetal calf serum. For small scale purification500 ml of conditioned medium from static flask or spinner cultures washarvested by centrifugation and 0.1 volumes of 1.0M TrisHCl pH8.0 and0.5 to 1.0 ml of Protein A-agarose (Boehringer Mannheim, Lewes, U.K.)were added. This was stirred overnight at room temperature thencollected on a disposable column. This was washed with 10 column volumesof 0.1M TrisHCl pH8.0, 10 column volumes of 0.01M TrisHCl pH8.0 andeluted with 0.1M glycine buffer, pH3.0. 1.0 ml fractions were collectedinto tubes containing 100 μl of 1.0M TrisHCl, pH8.0. Fractionscontaining antibody were pooled and dialysed against PBS. Theconcentrations of the antibody preparations were determined using aMicro BCA Protein Assay Reagent Kit (Pierce, Rockford, U.S.A.). Sampleswere checked by running on 10% SDS-polyacrylamide gels.

Additional changes to the variable region were introduced in order toimprove the affinity of the reshaped antibody, FB5HuVH/HuVK for FB5. Thechimeric FB5 antibody, in which the murine constant region domains ofthe heavy and light chains had been replaced by the human constantregions used in the humanized antibody, was constructed as described byOrlandi et al., (1989). Two hybrid chimeric/humanized antibodies wereconstructed consisting of the chimeric heavy chain with the humanizedlight chain and the humanized heavy chain with the chimeric light chain.

Both of these antibodies showed binding to the LA1-5s target cells withsimilar efficacies to the chimaeric and murine antibodies (within 3fold). Despite these efficacies, further attention was directed towardsthe heavy and light chains in order to improve the affinity of thehumanized antibody.

Three further versions of the FB5HuVH and the HuVK, were designed. Theamino acid sequences of these VHs and VKs are shown in table 1.

Table 1 shows the variable region sequences of FB5HuVH, FB5HuVHK,FB5HuVHQA,LT,VK, FB5HuVHK,Q,A,LT,VK, FB5HuVK and FB5HuVKF. Murineframework residues are shown in lower case. Residue REI L(104) andT(107) are unusual residues for human subgroup I kappa chains; theseresidues have been replaced by the more common, V and K residues(underlined in table 1).

The additional changes to the HuVH and HuVK constructs are shown below(numbering according to Kabat et al., ibid):

FB5HuVHK (38), FB5HuVHQA,LT,VK (66-67, 69-70, 72-73), FB5HuVHK,QA,LT,VK(38, 66-67, 69-70, 72-73), and FB5HuVKF (71). Two variations of theHuVKs shown in table 5 were constructed by inclusion of a Y residue atposition 36.

These new versions were constructed by mutagenesis of the originalreshaped heavy chain M13 single stranded DNA clone. The method ofHiguchi, R. et al. (1988) Nucleic Acids Res. 16, 7351-7367, whichutilizes overlapping PCR amplification with mutagenic primers, wasemployed. The modified variable regions were cloned into the expressionvector pSVgpt as before and cotransfected with the MuVK plasmid into NS0cells. Antibody producing cell clones were selected, expanded andpurified for testing. Subsequent to this, fully humanized versionantibodies consisting of the modified HuVHs and HuVKs were prepared inthe same way.

TABLE 1 FB5HuVH: QVQLQESGPGLVRPSQTLSLTCTaSGyTFtDYVIHWVRQPPGRGLEWIGYINPYDDDTTYNQKFKGRV TMLVDTSsNtayLRLSSVTAeDTAVYYCARRGNSYDGYFDYSMDYWGQGSLVTVSS (SEQ ID NO:16) FB5HuVHK:QVQLQESGPGLVRPSQTLSLTCTaSGyTYVI HWVkQPPGRGLEWIGYINPYDDDTTYNQKFKGRVTMLVDTSsNtayLRLSSVTAeDTAVYYCARRGNS YDGYFDYSMDYWGQGSLVTVSS (SEQ ID NO:17)FB5HuVHQA, LT, VK: QVQLQESGPGLVRPSQTLSLTCTaSGyTFtDYVIHWVRQPPGRGLEWIGYINPYDDDTTYNQKFKGqa TltVvkSsNtayLRLSSVTAeDTAVYYCARRGNSYDGYFDYSMDYWGQGSLVTVSS (SEQ ID NO:18) FB5HuVHK, QA, LT, VK:QVQLQESGPGLVRPSQTLSLTCTaSGyTFtDYVI HWVkQPPGRGLEWIGYINPYDDDTTYNQKFKGqaTltVvkSsNtayLRLSSVTAeDTAVYYCARRGNS YDGYFDYSMDYWGQGSLVTVSS (SEQ ID NO:19)FB5HuVK: DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWLQQTPGKAPKLLIYSASNRYTGVPSRFSGSGSG TDYTFTISSLQPEDIATYYCQQYTNYPMYTFGQGTKVQIK (SEQ ID NO:20) FB5HuVKF: DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWLQQTPGKAPKLLIYSASNRYTGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQYTNYPMYTFGQGTKVQIK (SEQ ID NO:21)

Example 2 Specific Binding of Humanized FB5 Antibodies to CarcinomaCells

Recombinant antibodies (chimaeric and humanized) were tested in mixedhemadsorption (MHA) resetting assays (Rettig et al. (1987) J. Immunol.138, 4484-4489; Rettig et al. (1985) Cancer Res. 45, 815-821) for theirability to bind to LA1-5s target cells (human neuroblastoma cells). Thecells were grown in 60 well Terasaki plates to form confluentmonolayers. Cells were washed twice with PBSB and 10 μl of antibodyadded to the cells (antibodies diluted in DMEM without fetal calf serum,serially diluted two fold. Incubation with test antibody was continuedat room temperature for 1 hour after which cells were washed three timeswith PBSB and incubated with human red blood cells (type O+) conjugatedto protein A (Pierce, Ill., U.S.A.) diluted in PBSB. Incubation with theindicator cells was continued at room temperature for 30 min after whichunbound PA-RBCs were removed by washing twice with PBSB. The percentageresetting for each dilution of antibody was determined and theconcentration of antibody required for 50% resetting calculated. Thisdata for the recombinant antibodies is presented below.

Concentration of antibody for Antibody 50% rosetting (ng/ml) Murine FB5 3 Chimaeric FB5  3 FB5HuVH/HuVK 80 FB5HuVHK/HuVK 16 FB5HuVHK, QA, LT,VK/HuVK 16

Appropriate negative controls were included in experiments

These and other recombinant antibodies have been tested in ELISAs usingthe LA1-5s target cells. The ELISA method used is as follows:

LA1-5s cells are diluted to 1.5×10⁵−2.5×10⁵ cells/ml in DMEM, 10% FCSand 200 μl (ie 3-5×10⁴ cells) added to each well. Cells are grown untilnearly confluent (about 2 days). Plates are washed 2×with PBS and 100 μlantibody (diluted in DMEM) added. Incubation is carried out at 4° C. for1 hour. The wells are washed 3×with PBS and 100 μl of appropriatereporter antibody added, ie goat anti-human IgGl, HRPO conjugate(Sera-lab, 0.4 mg/ml, diluted in 1: 500 in DMEM); incubation is carriedout at 4° C. for 1 hour. Wells are washed 3×with PBS and bound reporterantibody detected using H₂O₂ and o-phenylenediaminedihydrochloride andthe OD 492 nm measured.

The ELISA data presented in the attached graphs, together with theresetting data, indicate that humanized antibodies of the Examples bindthe FB5 antigen. In particular the FB5HuVHK/HuVK antibody exhibitsbinding affinities close to the murine and chimaeric FB5 antibodies.Such recombinant antibodies (of which these are examples) thereforeprovide for novel, recombinant antibody molecules for the diagnosis andtherapy of human cancers characterized by the expression of the FB5antigen in the tumor stroma.

INCORPORATION BY REFERENCE

All patents, patents applications, and publications cited areincorporated herein by reference.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Indeed, variousmodifications of the above-described makes for carrying out theinvention which are obvious to those skilled in the field of molecularbiology or related fields are intended to be within the scope of thefollowing claims.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 25(2) INFORMATION FOR SEQ ID NO:1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 31 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: unknown           (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GGAAGCTTAG ACCGATGGGG CTGTTGTTTT G         #                  #          31 (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 32 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: unknown          (D) TOPOLOGY: unknown     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:GGAAGCTTGA AGATGGATAC AGTTGGTGCA GC        #                  #          32 (2) INFORMATION FOR SEQ ID NO:3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 22 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: unknown          (D) TOPOLOGY: unknown     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:AGGTSMARCT GCAGSAGTCW GG            #                  #                 22 (2) INFORMATION FOR SEQ ID NO:4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 28 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: unknown          (D) TOPOLOGY: unknown     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:ACTAGTCGAC ATGGRCTTHM AGRTGSAG          #                  #             28 (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 75 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: unknown          (D) TOPOLOGY: unknown     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:CGTCCAGGTG GCTGTCTCAC CCAGTGTATA ACATAGTCAG TGAAGGTGTA GC#CAGACGCG     60 GTGCAGGTCA GGCTC               #                  #                   #    75 (2) INFORMATION FOR SEQ ID NO:6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 76 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: unknown          (D) TOPOLOGY: unknown     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:TTGTCACTCT GCCCTTGAAC TTCTGGTTGT AGGTAGTATC ATCATCATAA GG#ATTAATAT     60 ATCCAATCCA CTCAAG              #                  #                   #    76 (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 96 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: unknown          (D) TOPOLOGY: unknown     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:CCTGAGGAGA CGGTGACGAG ACTCCCTTGG CCCCAGTAGT CCATAGAGTA GT#CAAAGTAA     60 CCATCATAGG AATTCCCCCT TCTTGCACAA TAATAG      #                   #       96 (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 69 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: unknown          (D) TOPOLOGY: unknown     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:GGAGCCTTAC CTGGGGTCTG CTGGTACCAG GCTACAGCAG TACCCACATT CT#GGCTGGCT     60 CTACAGGTG                 #                  #                   #         69 (2) INFORMATION FOR SEQ ID NO:9:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 48 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: unknown          (D) TOPOLOGY: unknown     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:CTGCTTGGCA CACCAGTGTA CCGATTCGAT GCCGAGTAGA TCAGCAGC  #                48 (2) INFORMATION FOR SEQ ID NO:10:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 83 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: unknown          (D) TOPOLOGY: unknown     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:CTACTCACGT TTGATTTGCA CCTTGGTCCC TTGGCCGAAC GTGTACATGG GA#TAGTTGGT     60 ATATTGCTGG CAGTAGTAGG TGG           #                   #                83(2) INFORMATION FOR SEQ ID NO:11:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 64 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: unknown           (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:CGCGGTGTCC TCGGCTGTCA CGCTGCTGAG TCTCAGGTAG GCTGTGTTGG AG#CTGGTGTC     60 TACC                  #                  #                   #             64 (2) INFORMATION FOR SEQ ID NO:12:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 16 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: unknown          (D) TOPOLOGY: unknown     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:AACAGCTATG ACCATG              #                   #                  #    16 (2) INFORMATION FOR SEQ ID NO:13:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 22 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: unknown          (D) TOPOLOGY: unknown     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:CTCTCTCAGG GCCAGGCGGT GA            #                  #                 22 (2) INFORMATION FOR SEQ ID NO:14:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 17 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: unknown          (D) TOPOLOGY: unknown     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:GTAAAACGAC GGCCAGT              #                   #                  #   17 (2) INFORMATION FOR SEQ ID NO:15:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 22 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: unknown          (D) TOPOLOGY: unknown     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:GCGGGCCTCT TCGCTATTAC GC            #                  #                 22 (2) INFORMATION FOR SEQ ID NO:16:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 124 amino #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: unknown           (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Le #u Val Arg Pro Ser Gln1               5    #                10   #                15Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Ty #r Thr Phe Thr Asp Tyr            20       #            25       #            30Val Ile His Trp Val Arg Gln Pro Pro Gly Ar #g Gly Leu Glu Trp Ile        35           #        40           #        45Gly Tyr Ile Asn Pro Tyr Asp Asp Asp Thr Th #r Tyr Asn Gln Lys Phe    50               #    55               #    60Lys Gly Arg Val Thr Met Leu Val Asp Thr Se #r Ser Asn Thr Ala Tyr65                   #70                   #75                   #80Leu Arg Leu Ser Ser Val Thr Ala Glu Asp Th #r Ala Val Tyr Tyr Cys                85   #                90   #                95Ala Arg Arg Gly Asn Ser Tyr Asp Gly Tyr Ph #e Asp Tyr Ser Met Asp            100       #           105       #           110Tyr Trp Gly Gln Gly Ser Leu Val Thr Val Se #r Ser         115          #       120 (2) INFORMATION FOR SEQ ID NO:17:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 124 amino #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: unknown           (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Le #u Val Arg Pro Ser Gln1               5    #                10   #                15Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Ty #r Thr Phe Thr Asp Tyr            20       #            25       #            30Val Ile His Trp Val Lys Gln Pro Pro Gly Ar #g Gly Leu Glu Trp Ile        35           #        40           #        45Gly Tyr Ile Asn Pro Tyr Asp Asp Asp Thr Th #r Tyr Asn Gln Lys Phe    50               #    55               #    60Lys Gly Arg Val Thr Met Leu Val Asp Thr Se #r Ser Asn Thr Ala Tyr65                   #70                   #75                   #80Leu Arg Leu Ser Ser Val Thr Ala Glu Asp Th #r Ala Val Tyr Tyr Cys                85   #                90   #                95Ala Arg Arg Gly Asn Ser Tyr Asp Gly Tyr Ph #e Asp Tyr Ser Met Asp            100       #           105       #           110Tyr Trp Gly Gln Gly Ser Leu Val Thr Val Se #r Ser         115          #       120 (2) INFORMATION FOR SEQ ID NO:18:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 124 amino #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: unknown           (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE:     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Le #u Val Arg Pro Ser Gln1               5    #                10   #                15Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Ty #r Thr Phe Thr Asp Tyr            20       #            25       #            30Val Ile His Trp Val Arg Gln Pro Pro Gly Ar #g Gly Leu Glu Trp Ile        35           #        40           #        45Gly Tyr Ile Asn Pro Tyr Asp Asp Asp Thr Th #r Tyr Asn Gln Lys Phe    50               #    55               #    60Lys Gly Gln Ala Thr Leu Thr Val Val Lys Se #r Ser Asn Thr Ala Tyr65                   #70                   #75                   #80Leu Arg Leu Ser Ser Val Thr Ala Glu Asp Th #r Ala Val Tyr Tyr Cys                85   #                90   #                95Ala Arg Arg Gly Asn Ser Tyr Asp Gly Tyr Ph #e Asp Tyr Ser Met Asp            100       #           105       #           110Tyr Trp Gly Gln Gly Ser Leu Val Thr Val Se #r Ser         115          #       120 (2) INFORMATION FOR SEQ ID NO:19:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 124 amino #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: unknown           (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Le #u Val Arg Pro Ser Gln1               5    #                10   #                15Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Ty #r Thr Phe Thr Asp Tyr            20       #            25       #            30Val Ile His Trp Val Lys Gln Pro Pro Gly Ar #g Gly Leu Glu Trp Ile        35           #        40           #        45Gly Tyr Ile Asn Pro Tyr Asp Asp Asp Thr Th #r Tyr Asn Gln Lys Phe    50               #    55               #    60Lys Gly Gln Ala Thr Leu Thr Val Val Lys Se #r Ser Asn Thr Ala Tyr65                   #70                   #75                   #80Leu Arg Leu Ser Ser Val Thr Ala Glu Asp Th #r Ala Val Tyr Tyr Cys                85   #                90   #                95Ala Arg Arg Gly Asn Ser Tyr Asp Gly Tyr Ph #e Asp Tyr Ser Met Asp            100       #           105       #           110Tyr Trp Gly Gln Gly Ser Leu Val Thr Val Se #r Ser         115          #       120 (2) INFORMATION FOR SEQ ID NO:20:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 108 amino #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: unknown           (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Le #u Ser Ala Ser Val Gly1               5    #                10   #                15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gl #n Asn Val Gly Thr Ala            20       #            25       #            30Val Ala Trp Leu Gln Gln Thr Pro Gly Lys Al #a Pro Lys Leu Leu Ile        35           #        40           #        45Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pr #o Ser Arg Phe Ser Gly    50               #    55               #    60Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Il #e Ser Ser Leu Gln Pro65                   #70                   #75                   #80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ty #r Thr Asn Tyr Pro Met                85   #                90   #                95Tyr Thr Phe Gly Gln Gly Thr Lys Val Gln Il #e Lys             100      #           105 (2) INFORMATION FOR SEQ ID NO:21:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 108 amino #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: unknown           (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Le #u Ser Ala Ser Val Gly1               5    #                10   #                15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gl #n Asn Val Gly Thr Ala            20       #            25       #            30Val Ala Trp Leu Gln Gln Thr Pro Gly Lys Al #a Pro Lys Leu Leu Ile        35           #        40           #        45Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pr #o Ser Arg Phe Ser Gly    50               #    55               #    60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Il #e Ser Ser Leu Gln Pro65                   #70                   #75                   #80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ty #r Thr Asn Tyr Pro Met                85   #                90   #                95Tyr Thr Phe Gly Gln Gly Thr Lys Val Gln Il #e Lys             100      #           105 (2) INFORMATION FOR SEQ ID NO:22:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 372 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: unknown           (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:CAGGTSMARC TGCAGSAGTC WGGACCTGAG CTGGTGAAGC CTGGGGCTTC AG#TGAAGATG     60TCCTGCAAGG CTTCTGGATA CACATTCACT GACTATGTTA TACACTGGAT GA#AGCAGAGA    120AATGGAAAGA GCCTTGAGTG GATTGGATAT ATTAATCCTT ATGATGATGA TA#CTACCTAC    180AACCAGAAGT TCAAGGGCCA GGCCACATTG ACTGTAGTCA AATCCTCCAA CA#CAGCCTAC    240ATGCAGCTCA ACAGCCTGAC ATCTGAGGAC TCTGCAGTCT ATTACTGTGC AA#GAAGGGGG    300AATTCCTATG ATGGTTACTT CGACTATTCT ATGGACTACT GGGGTCAAGG AA#CCTCAGTC    360 ACCGTCTCCT CA               #                  #                   #      372 (2) INFORMATION FOR SEQ ID NO:23:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 124 amino #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: unknown           (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein     (ix) FEATURE:          (A) NAME/KEY: Xaa           (B) LOCATION: 3..4          (D) OTHER INFORMATION:  #Xaa = Lys or Glu     (ix) FEATURE:          (A) NAME/KEY: Xaa           (B) LOCATION: 6..7          (D) OTHER INFORMATION:  #Xaa = Glu or Gln    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:Gln Val Xaa Leu Gln Xaa Ser Gly Pro Glu Le #u Val Lys Pro Gly Ala1               5    #                10   #                15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Ty #r Thr Phe Thr Asp Tyr            20       #            25       #            30Val Ile His Trp Met Lys Gln Arg Asn Gly Ly #s Ser Leu Glu Trp Ile        35           #        40           #        45Gly Tyr Ile Asn Pro Tyr Asp Asp Asp Thr Th #r Tyr Asn Gln Lys Phe    50               #    55               #    60Lys Gly Gln Ala Thr Leu Thr Val Val Lys Se #r Ser Asn Thr Ala Tyr65                   #70                   #75                   #80Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Se #r Ala Val Tyr Tyr Cys                85   #                90   #                95Ala Arg Arg Gly Asn Ser Tyr Asp Gly Tyr Ph #e Asp Tyr Ser Met Asp            100       #           105       #           110Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Se #r Ser         115          #       120 (2) INFORMATION FOR SEQ ID NO:24:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 325 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: unknown           (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:GACATTGTGA TGACCCAGTC TCAAAAATTC ATGTCCACAT CAGTAGGAGA CC#AGGGTCAA     60CATCACCTGC AGGGCCAGTC AGAATGTGGG TACTGCTGTA GCCTGGTATC AA#CAGAAACC    120AGGACAATCT CCTAAATTAC TGATTTACTC GGCATCGAAT CGGTACACTG GA#GTCCCTGA    180TCGCTTCACA GGCAGTGGAT CTGGGACAGA TTTCACTCTC ACCATCAGCA AT#ATGCAGTC    240TGAAGACCTG GCAGATTATT TCTGCCAGCA ATATACCAAC TATCCCATGT AC#ACGTTTGG    300 AGGGGGGACC AAGCTGGAAA TAAAA          #                   #              325 (2) INFORMATION FOR SEQ ID NO:25:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 108 amino #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: unknown           (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:Asp Ile Val Met Thr Gln Ser Gln Lys Phe Me #t Ser Thr Ser Val Gly1               5    #                10   #                15Asp Arg Val Asn Ile Thr Cys Thr Ala Ser Gl #n Asn Val Gly Thr Ala            20       #            25       #            30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Se #r Pro Lys Leu Leu Ile        35           #        40           #        45Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pr #o Asp Arg Phe Thr Gly    50               #    55               #    60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Il #e Ser Asn Met Gln Ser65                   #70                   #75                   #80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Ty #r Thr Asn Tyr Pro Met                85   #                90   #                95Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Il #e Lys             100      #           105

What is claimed is:
 1. A method for detecting presence of cancer cellsin a sample comprising contacting said sample with a humanized antibodywhich specifically binds to an FB5 antigen, comprising a humanizedvariable region having a heavy chain region and a light chain region,said heavy chain region having an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 16, 17, 18, and 19, and said light chainregion having an amino acid sequence selected from the group consistingof SEQ ID NOS: 20 and 21, and determining binding of said humanizedantibody to FB5 antigen in said sample as a determination of cancercells in said sample.
 2. The method of claim 1, wherein said humanizedantibody is labelled.
 3. The method of claim 2, wherein said humanizedantibody is labelled with an enzyme.
 4. The method of claim 1, whereinsaid sample is taken from a human.
 5. The method according to claim 1,wherein the heavy chain region has the amino acid sequence of SEQ ID NO:16 and the light chain region has the amino acid sequence of SEQ ID NO:20.
 6. The method according to claim 1, wherein the heavy chain regionhas the amino acid sequence of SEQ ID NO: 17 and the light chain regionhas the amino acid sequence of SEQ ID NO:
 20. 7. The method according toclaim 1, wherein the heavy chain region has the amino acid sequence ofSEQ ID NO: 18 and the light chain region has the amino acid sequence ofSEQ ID NO:
 20. 8. The method according to claim 1, wherein the heavychain region has the amino acid sequence of SEQ ID NO: 19 and the lightchain region has the amino acid sequence of SEQ ID NO:
 20. 9. The methodaccording to claim 1, wherein the heavy chain region has the amino acidsequence of SEQ ID NO: 16 and the light chain region has the amino acidsequence of SEQ ID NO:21.
 10. The method according to claim 1, whereinthe heavy chain region has the amino acid sequence of SEQ ID NO: 17 andthe light chain region has the amino acid sequence of SEQ ID NO:
 21. 11.The method according to claim 1, wherein the heavy chain region has theamino acid sequence of SEQ ID NO: 18 and the light chain region has theamino acid sequence of SEQ ID NO:
 21. 12. The method according to claim1, wherein the heavy chain region has the amino acid sequence of SEQ IDNO: 19 and the light chain region has the amino acid sequence of SEQ IDNO:
 21. 13. The method of claim 2, wherein said humanized antibody islabelled with a radionuclide, a fluorescent chromophore, an enzyme, oran electron dense molecule.
 14. The method of claim 13, wherein saidradionuclide is ¹²⁵I, ¹³¹I, or ¹⁴C.
 15. The method of claim 13, whereinsaid fluorescent chromophore is fluorescein, phycobiliprotein ortetraethyl rhodamine.
 16. The method of claim 13, wherein said enzyme isperoxidase.
 17. The method of claim 13, wherein said electron densemolecule is ferritin or gold.
 18. A method for determining presence ofcancer in a subject, comprising administering to said subject an amountof a humanized antibody which specifically binds to an FB5 antigensufficient to bind to said FB5 antigen and indicate presence of saidantigen in said subject, said humanized antibody comprising a humanizedvariable region having a heavy chain region and a light chain region,said heavy chain region having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 16, 17, 18, and 19, and said light chainhaving an amino acid sequence selected from the group consisting of SEQID NOS: 20 and 21, wherein binding of said humanized antibody to FB5antigen is indicative of cancer in said subject.
 19. The method of claim18, wherein said subject is a human.
 20. The method of claim 18, whereinsaid humanized antibody is labelled.
 21. The method of claim 20, whereinsaid antibody is labeled with a radionuclide or with a fluorescentchromophore.
 22. The method of claim 21, wherein said radionuclide is¹²⁵I or ¹³¹I.
 23. The method of claim 21, wherein said fluorescentchromophore is fluorescein, phycobiliprotein or tetraethyl rhodamine.24. The method of claim 18, wherein the heavy chain region has the aminoacid sequence of SEQ ID NO: 16 and the light chain region has the aminoacid sequence of SEQ ID NO:
 20. 25. The method of claim 18, wherein theheavy chain region has the amino acid sequence of SEQ ID NO: 17 and thelight chain region has the amino acid sequence of SEQ ID NO:
 20. 26. Themethod of claim 18, wherein the heavy chain region has the amino acidsequence of SEQ ID NO: 19 and the light chain region has the amino acidsequence of SEQ ID NO:
 20. 27. The method of claim 18, wherein the heavychain region has the amino acid sequence of SEQ ID NO: 16 and the lightchain region has the amino acid sequence of SEQ ID NO:
 21. 28. Themethod of claim 18, wherein the heavy chain region has the amino acidsequence of SEQ ID NO: 17 and the light chain region has the amino acidsequence of SEQ ID NO:
 21. 29. The method of claim 18, wherein the heavychain region has the amino acid sequence of SEQ ID NO: 18 and the lightchain region has the amino acid sequence of SEQ ID NO:
 21. 30. Themethod of claim 18, wherein the heavy chain region has the amino acidsequence of SEQ ID NO: 19 and the light chain region has the amino acidsequence of SEQ ID NO:
 21. 31. The method of claim 18, wherein the heavychain region has the amino acid sequence of SEQ ID NO: 18 and the lightchain region has the amino acid sequence of SEQ ID NO:20.